“Endotoxin” is a component of the cell wall in the outer membrane of gram-negative bacteria, and its activity is mainly attributed to LPS (lipopolysaccharide). In the living body, endotoxin exists as a part of the outer membrane in the surface layer of gram negative bacteria. Generally, after death of gram-negative bacteria, endotoxin is liberated and is present in a free form in blood.
When more than a certain level of endotoxin is present in blood, the endotoxin stimulates monocytes, granulocytes, etc., resulting in excessive production of inflammatory cytokines. Consequently, so called endotoxinemia accompanied by symptoms such as fever, sepsis, septic shock, multiple organ failure, etc. is induced. For this reason, detection of endotoxin in pharmaceuticals for injection, etc. is crucial, and thus the bacterial endotoxin test is prescribed by the Japanese, U.S. and European pharmacopeias. From the aspect of clinical diagnosis, precise measurement of blood endotoxin level is considered crucial for early diagnosis and therapeutic effect evaluation.
Examples of a conventional method for measuring endotoxin include the pyrogen test, in which a rabbit is treated with a direct injection of a test sample and measured for increase in body temperature that can be converted into the endotoxin level, and the Limulus test utilizing gelation of horseshoe crab amebocyte lysate triggered by endotoxin. The method involving direct injection into a rabbit has problems in cost, length of time required to obtain the test results, and sensitivity, and for this reason, the Limulus test currently prevails as a method for measuring endotoxin.
A gelation process of horseshoe crab amebocyte lysate triggered by endotoxin is shown in FIG. 1. The gelation process of horseshoe crab amebocyte lysate contains a Factor C pathway specifically associated with endotoxin. The “Factor C pathway” is constituted by the following cascades. First, endotoxin firmly binds with Factor C, and thereby activates the Factor C. Then, Factor C activated by binding with endotoxin (active Factor C) activates Factor B. Subsequently, activated Factor B (active Factor B) activates a proclotting enzyme, resulting in production of a clotting enzyme. This clotting enzyme partially hydrolyzes its substrate, i.e., coagulogen. As a result, peptide C is liberated from the coagulogen, and a clotting protein, coagulin, is produced. By a coagulation action of the coagulin, gelation occurs (see nonpatent literature 1).
The Limulus test for measuring endotoxin utilizes the above-mentioned gelation process of horseshoe crab amebocyte lysate triggered by endotoxin. As the Limulus test, a gel-clot technique, a colorimetric technique using synthetic chromogenic substrates, a kinetic turbidimetric technique and the like are known and they are different in the ways of evaluation and measurement (see nonpatent literature 2).
“The gel-clot technique” is a method involving mixing horseshoe crab amebocyte lysate with a test sample in a test tube,
allowing the mixture to react under certain conditions (for example, 37° C. for 30 to 60 minutes),
inverting or tilting the test tube, and then
evaluating the test sample based on whether the mixture remains liquid or is solidified.
The test sample is evaluated as endotoxin negative in the former case, and as endotoxin positive in the latter case. This technique can be performed in a relatively easy manner without need of any special device. The technique, however, has problems in that difference of the material and production lots easily affects the test results, and in that the results are less objective as compared with optical methods because the way of evaluation relies on human eye observation. Therefore, the technique is usually used just as a quick test.
In the “colorimetric technique using synthetic chromogenic substrates,” a synthetic chromogenic peptide substrate is used as a substrate of the clotting enzyme. The technique involves calculating an endotoxin level from absorbance of the amount of a released chromophore by colorimetric quantification. The synthetic chromogenic peptide substrate to be used has an amino acid sequence similar to that of the clotting enzyme-mediated hydrolysis site of a natural substrate coagulogen. The cleavage site (hydrolysis site) is bound to a chromophore, such as para-nitroaniline (pNA), and when the clotting enzyme cleaves off this chromophore, the free chromophore causes color formation. When the chromophore is para-nitroaniline, the absorbance at 405 nm, which is the maximal absorption wavelength of para-nitroaniline, is measured with time. When the chromophore is a diazotized para-nitroaniline, the absorbance at 545 nm is measured with time. After that, the resulting change with time in transmitted light amount is analyzed to determine the endotoxin level. The colorimetric technique using synthetic chromogenic substrates has problems, for example, in that reagents are relatively expensive, and in that the procedures are complicated, but is excellent in quantitative reliability, sensitivity and objectivity.
In the “kinetic turbidimetric technique,” increase in turbidity caused by gelation is observed as change in transmitted light amount, and the elapsed time required for the transmitted light through the reaction mixture to decrease in ratio to a given threshold (usually about 90%) is regarded as a gelation time. The technique involves calculating an endotoxin level by use of a standard curve obtained by plotting the gelation time against the endotoxin level. This technique is extremely excellent in quantitative reliability and objectivity, but needs a dedicated device for measurement.
A reagent kit that enables highly sensitive measurement of endotoxin using a recombinant Factor C and a fluorescent substrate (trade name: PyroGene rFc, manufacturer: Lonza Walkersville, Inc., distributor: Daiichi Pure Chemicals Co., Ltd.) is commercially available.